Original Paper Published online: June 6, 2015
Pharmacology 2015;96:25–31 DOI: 10.1159/000431100
Ginsenoside Rg1 Ameliorates Motor Function in an Animal Model of Parkinson’s Disease Wenda Jiang a Zhejian Wang b Yiyang Jiang a Meili Lu c Xin Li d
a
Department of Neurology, First Affiliated Hospital of Liaoning Medical University, Jinzhou, b Dalian Medical University, Dalian, c Institute of Medicine, Liaoning Medical University, Jinzhou, d Department of Neurology, Jinzhou Central Hospital, Jinzhou, China
Key Words Parkinson’s disease · Ginsenoside Rg1 · Dopaminergic neuron · MPTP
significantly rescue the deficit of motor function in mice model of PD, and suggest that Rg1 may be a potential therapeutic agent against PD and related disorders. © 2015 S. Karger AG, Basel
© 2015 S. Karger AG, Basel 0031–7012/15/0962–0025$39.50/0 E-Mail [email protected] www.karger.com/pha
Introduction
Parkinson’s disease (PD) is one of the major neurodegenerative disorders, which is characterized by disabling motor abnormalities including tremor, muscle stiffness, paucity of voluntary movements and postural instability [1–3]. The main neuropathological feature of PD is the loss of dopaminergic neurons in the substantia nigra (SN) and diminished dopamine levels in the striatum. Accumulating evidence supports that nigral neuronal loss in PD is at least in part due to the aggregation of α-synuclein protein in the cytoplasm [4, 5]. Indeed, genetic studies have shown that α-synuclein knockdown by siRNAs protects dopaminergic neurons from MPTP-induced cell death both in vitro and in vivo [6–9]. Ginseng has been used in traditional Chinese medicine to enhance stamina and capacity to deal with fatigue and physical stress for thousands of years. Ginsenoside Rg1 is the major active ingredient of ginseng. It has been widely reported that Rg1 has a wide range of neurotrophic Wenda Jiang, MD Department of Neurology First Affiliated Hospital of Liaoning Medical University 5-2 Renmin Street, Jinzhou 121000 (China) E-Mail jiang_neurology @ 163.com
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Abstract Aims: Parkinson’s disease (PD) is a neurodegenerative disorder characterized by loss of dopaminergic neurons in the substantia nigra (SN) and diminished dopamine levels in the striatum. Accumulating evidence supported that ginsenoside Rg1, the major pharmacologically active compound of ginseng, has a wide range of neurotrophic and neuroprotective effects under physiological and pathological conditions. Although Rg1 administration protects dopaminergic neurons in a rat model of PD, it is unclear if Rg1 treatment ameliorates motor function in PD. Methods: Using the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) that causes dopaminergic neurodegeneration, we investigated the effect of Rg1 on 3 tests of motor behaviors in mice: the accelerating rotarod, wire suspension and pole tests. Results: The results showed that Rg1 treatment (10 mg/kg, i.p.) succeeded in restoring motor functions to physiological level in MPTP-treated mice. Importantly, these behavioral ameliorations were accompanied by an attenuation of the MPTP-induced loss of dopaminergic neurons in the SN and striatum. Conclusions: These findings indicate that Rg1 can
Rg1 injection
Fig. 1. Experimental design. Mice received
and neuroprotective effects both in vitro and in vivo. For example, in vitro studies have showed that Rg1 protects dopaminergic cells against glutamate [10], MPTP [11, 12] and rotenone toxicities [13]. In vivo studies have also demonstrated the protective effect of Rg1 against MPTPinduced nigral neuronal loss [14]. However, it is undetermined if Rg1 treatment ameliorates motor function in MPTP-treated model of PD. Since a growing body of evidence has showed that Rg1 has obviously protective effects on dopaminergic neurons, we hypothesize that Rg1 treatment may ameliorate motor function. In this study, we investigated this hypothesis using a combination of behavioral, immunoblotting and immunohistochemical assessments in MPTP-treated mice. Materials and Methods Animals and Treatment C57BL/6 mice (25–30 g, purchased from Charles River (Beijing Office, China)) were housed in plastic cages with free access to food and water and maintained in a temperature-controlled room (21 ° C) with a 12/12 h light/dark cycle. All behavioral experiments were performed according to the guidelines of Liaoning Medical University Animal House Center and every effort was made to minimize both the animal suffering and the number of animals used. Mice received intraperitoneal (i.p.) injection of 30 mg/kg parkinsonian toxin MPTP hydrochloride once a day for 5 days to induce dopaminergic neuron death in the SN, while the control mice received equal amount of saline injection. Rg1 (10 mg/kg, i.p.) was injected into the MPTP-treated mice once a day from the first day of MPTP injection until 10 days after the last injection of MPTP. The experimental design is described in fig. 1.
Chemicals and Reagents Anti-α-synuclein and anti-tyrosine hydroxylase (TH) antibody are obtained from BD Transduction Laboratories. Anti-β-actin antibody is obtained from Abcam. Rg1 and MPTP hydrochloride are obtained from Sigma. Rotarod Test One week after the last MPTP injection, mice performances on the rod were evaluated on the accelerating rotarod apparatus (Stoelting Co.). The test consisted of a suspended rod (diameter:
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Pharmacology 2015;96:25–31 DOI: 10.1159/000431100
MPTP injection
1 2 3 4 5
13 14 15 Behavioral test
3 cm) that accelerates at a constant rate, from 4 to 40 rpm in 300 s. Mice were tested 3 consecutive days and at each day of test, they were placed on the rod for a session of 10 trials at 3 min intervals, and the time that they remained on the rotarod during each trial was recorded. The maximum test time (cut-off limit) was 300 s. Pole Test The pole test was modified from previously reported protocols [15]. The pole consisted of a thin wooden cylinder (length: 50 cm, diameter: 1.5 cm) and a cross-shaped wooden base placed in a clean cage. Rubber bands were wrapped around the cylinder at intervals of approximately 1.5 inches to increase traction. Mice were acclimated to the testing room for 1 h before each session. Mice were placed at the top of the pole facing downwards and latency to descend the pole was measured. Trials were excluded if the mouse jumped or slid down the pole rather than climbed down. Mice were pretrained 2 consecutive times at the first day of test and the test was performed at days 1, 2 and 3 before the rotarod test. Wire Suspension Test A wire (length: 80 cm, height: 25 cm) was fixed horizontally between two platforms. Each animal was hung with its paws from the middle of the wire, and the time needed to reach one platform was recorded. The maximal time allowed was set at 120 s. Performance was conducted at days 1, 2 and 3 before the initiation of the rotarod test. Immunoblotting Brain tissues were lysed on ice in the lysis buffer and then the solution was centrifuged at 14,000 rpm for 10 min at 4 ° C. Supernatant was collected and protein concentration was determined by BCA protein assay kit (Thermo Scientific). An equal amount of protein samples were mixed with 4× sample buffer, boiled at 100 ° C for 5 min, and separated on 10% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Proteins were then transferred to Immobilon-PTM polyvynilidene fluoride (PVDF) membranes (Bio-Rad). The membranes were blocked with 5% non-fat milk in Tris-buffered saline containing 0.1% Tween-20 (TBST) for 1 h at room temperature, and then incubated overnight at 4 ° C with primary antibody. After washing 3 × 5 min in TBST, membranes were incubated with horseradish peroxidase-conjugated secondary antibody for 1 h at room temperature. After another three washes with TBST, protein was visualized in the BioRad Imager using ECL Western blotting substrate (Pierce). The band density of each protein was quantified by the Bio-Rad Quantity One software and the relative optical density was analyzed relative to loading control beta-actin on the same membrane.
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a chronic MPTP treatment consisting of 1 injection (i.p.) of 30 mg/kg/day or the same volume of sterile saline for 5 days, and Rg1 (10 mg/kg, i.p.) was administrated daily throughout the experiment. Seven days after the last injection of MPTP, behavioral tests were performed for 3 days, and brain tissues were collected immediately after behavioral tests.
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Fig. 2. Behavioral tests. Effects of Rg1 treatment on MPTP-induced motor deficits in the paradigms of the rotarod test (a), pole test (b) and wire suspension test (c). Statistical analyses were car-
ried out using the two-way ANOVA, Tukey’s post hoc analysis. # p < 0.05, ## p < 0.01 vs. respective first day; * p < 0.05 vs. respective saline-treated mice. n = 10 in each group.
Immunohistochemistry Mice were anesthetized with 1.5 g/kg urethane (Sigma) and then perfused with 0.9% saline and 4% paraformaldehyde. The brain was taken out from the mice and fixed in 4% paraformaldehyde for one more day before they were transferred to 30% sucrose/PBS solution for cryoprotection. After the brain slices had sunk to the bottom of the sucrose solution, they were sectioned into 30 μm slices using Leica cryostat. After blocking and permeabilization using PBS solution containing 1% BSA and 0.2% Triton X-100 for 30 min at room temperature, the brain slices were incubated with anti-tyrosine hydroxylase antibody (1: 800 dilution) for 3 days at 4 ° C. Finally, the tyrosine hydroxylase-positive neurons on the slices were stained and visualized under light microscope using the anti-mouse Ig HRP detection kit (BD Transduction Laboratories, 551011) according to the manufacturer’s instruction.
(fig. 2a), indicating an impairment of motor balance and coordination. Importantly, daily Rg1 treatment fully rescued the MPTP-induced motor deficit, as reflected by an increase in the latency to fall off on test days 2 and 3 (fig. 2a). To further investigate the influence of Rg1 on motor capacities of MPTP-treated mice, we next introduced another two behavioral tasks, pole test and wire suspension test. Pole test estimated bradykinesia and motor coordination, whereas wire suspension test analyzed the coordination and muscle strength of mice [16, 17]. The results from pole test showed that mice treated with MPTP spent much more time descending the pole compared with those treated with saline (fig. 2b). As expected, Rg1 treatment succeeded in rescuing the motor deficit induced by MPTP, as reflected by a similar time in descending the pole compared with saline control (fig. 2b). Similarly, in comparison with saline controls, mice treated with MPTP spent much more time reaching the platform during the wire suspension test (fig. 2c), and Rg1 treatment restored the motor capacity to control levels (fig. 2c). Taken together, these results indicate that MPTP treatment produces Parkinson-like motor deficits that are frequently observed in patients with PD, and chronic Rg1 treatment can dramatically rescues these deficits.
Statistical Analysis Animals were randomly chosen for experiments and the data were analyzed by SPSS 13.0 and expressed as mean ± SEM. The differences of motor performance among different groups of mice were analyzed by two-way ANOVA, followed by Tukey’s post hoc test. The data of all other experiments were analyzed by one-way ANOVA, followed by Tukey’s post hoc test. * p < 0.05 and ** p < 0.01 were considered significant differences.
Results
Rg1 Ameliorates MPTP-Induced Motor Dysfunction Previous studies have shown that a core symptom of PD is motor abnormality [1–3]. To determine whether Rg1 can rescue MPTP-induced motor deficit, we first used a rotarod test to evaluate the motor balance in mice treated with Rg1. The results showed that the mice treated with MPTP spent significantly less time on the rod on test days 2 and 3 compared with those treated with saline Rg1 Treats Parkinson’s Disease
Rg1 Attenuates MPTP-Induced Loss of Dopaminergic Neurons in the SN and Striatum It is well documented that MPTP treatment produces loss of dopaminergic neurons in both SN and striatum [18–20]. To evaluate the protective effect of Rg1 on dopaminergic neurons, we examined the level of TH that is Pharmacology 2015;96:25–31 DOI: 10.1159/000431100
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Latency to fall (s)
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known to correlate with the level of dopaminergic neurons [21, 22]. As revealed by previous reports [18–20], we observed that TH expression significantly decreased in mice treated with MPTP in both SN (fig. 3a and b) and striatum (67.6 ± 10.1% saline, p < 0.05; fig. 3d and e) compared with saline control. Consistent with a recent report that Rg1 attenuates MPTP-induced apoptosis in mouse SN neurons [14], we found that chronic Rg1 treatment dramatically displayed the protective effect on loss of dopaminergic neurons in both SN (fig. 3a and b) and striatum (fig. 3d and e). Since previous reports have supported that loss of dopaminergic neurons in PD is at least in part due to the aggregation of α-synuclein protein in the cytoplasm [4, 5], we next examined if Rg1 treatment resulted in the degradation of α-synuclein in both SN and striatum. The result showed that MPTP treatment inPharmacology 2015;96:25–31 DOI: 10.1159/000431100
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duced a significant increase in α-synuclein expression in both SN (fig. 3a and c) and striatum (fig. 3d and f) compared with saline control. As expected, Rg1 treatment succeeded in restoring the α-synuclein expression to control level in both SN (fig. 3a and c) and striatum (fig. 3d and f). In order to further evaluate the protective effect of Rg1 on dopaminergic neurons, we counted TH-positive neurons in both SN and striatum. As showed in figure 4, dopaminergic neurodegeneration occurred morphologically after MPTP treatment. The numbers of TH-positive neurons significantly decreased in both SN (fig. 4a) and striatum (fig. 4b) compared with saline control. Similar to the results shown in figure 3, Rg1 treatment rescued the loss of dopaminergic neurons in both SN (fig. 4a) and striatum (fig. 4b). Jiang/Wang/Jiang/Lu/Li
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Fig. 3. Expression levels of TH and α-synuclein proteins were assessed by the Western blotting assay. a–c Immunoblotting for TH and α-synuclein in the SN. d–f Immunoblotting for TH and α-synuclein in the striatum. Statistical analyses were carried out using the one-way ANOVA, Tukey’s post hoc analysis. * p < 0.05 vs. saline-treated mice. n = 5 in each group.
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Discussion
In this study, we confirmed that ginsenoside Rg1, one of the active ingredients of ginseng, reduced the loss of dopaminergic neurons in both SN and striatum, and demonstrated that chronic Rg1 treatment ameliorated motor function and increased α-synuclein degradation in the MPTP-induced mice model of PD. Together, these results indicated that the neuroprotective mechanism of Rg1 may be attributed to an increase in the degradation of α-synuclein and subsequently to the prevention of MPTP-induced loss of dopaminergic neurons in SN and striatum. We have therefore provided evidence that Rg1 may be a potential therapeutic agent against PD via degrading increased α-synuclein in the SN and striatum. Rg1 Treats Parkinson’s Disease
0.3
The pathological hallmark of PD is the loss of dopaminergic neurons in the SN and diminished dopamine level in the striatum. Consistent with previous studies [18], we here found that dopaminergic neurons dramatically reduced in both SN and striatum in the MPTP-induced mice model of PD. But molecular mechanism underlying neuronal loss is not clear. Recent studies have shown that the abnormal accumulation of α-synuclein in the brain is a common feature of PD [23], and this may contribute to nigral neuronal loss in PD [4, 5]. Thus, it is reasonable to propose that the degradation of MPTP-induced α-synuclein may reduce the loss of dopaminergic neurons and subsequently rescue motor deficits in the MPTP-induced mice model of PD. Indeed, this hypothesis is supported by some previous genetic studies. For instance, knockdown α-synuclein by siRNAs protects dopaminergic Pharmacology 2015;96:25–31 DOI: 10.1159/000431100
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sentative images (left panel) and bar graph (right panel) show the influence of Rg1 on MPTP-induced the loss of TH-positive neurons in the SN. b Representative images (left panel) and bar graph (right panel) show the influence of Rg1 on MPTP-induced the loss of striatal fibers. Statistical analyses were carried out using the oneway ANOVA, Tukey’s post hoc analysis. * p < 0.05, ** p < 0.01 vs. saline-treated mice. n = 8 in each group.
MPTP
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0.4
neurons from MPTP-induced cell death both in vitro and in vivo [6–9]. In full agreement with these findings, we here reported that chronic Rg1 obviously increased the degradation of α-synuclein, and reduced the loss of dopaminergic neurons in the SN and striatum as well as rescued motor deficits. However, the involvement of Rg1 in α-synuclein degradation has been challenged, and a recent report indicates that only Rb1, but not Rg1 and Rg3, exhibits an obvious ability to disaggregate preformed fibrils of α-synuclein [24]. Discrepancies between the previous study and the present study still need to be resolved but may be at least in part accounted for by different experimental conditions, such as in vitro or in vivo. Alternatively, the neuroprotective effect of Rg1 against MPTP-induced neuronal loss may be related to the enhanced expression of Bcl-2 and reduced expression of nitric oxide synthase (NOS). It has been reported that Bcl-2 protects cells from apoptosis induced by exogenous oxidants or conditions that increase their intracellular production and at the same time reduce oxidative damage to cellular constituents [25–27]. Recent reports further confirmed the view that Rg1 partially overcame the decrease of Bcl-2 protein and gene expressions induced by 6-OHDA [28] or MPTP [14], therefore positively regulating cell survival. However, the exact pathway leading to the changes of Bcl-2 and NOS expression in the SN and
striatum is still unclear. Thus, future experiments determining the Bcl-2- and/or NOS-related signal pathways affected by Rg1 will be helpful to clinical application of Rg1 in treatment with PD and related disorders.
Conclusions
In summary, chronic ginsenoside Rg1 treatment reduces the loss of dopaminergic neurons in both SN and striatum and ameliorates motor function in the MPTPinduced mice model of PD. These ameliorations were accompanied by an increase in α-synuclein degradation. Thus, our findings indicate that Rg1 may be a potential therapeutic agent against PD in the future.
Acknowledgments W.J. designed research; W.J., Z.W., Y.J., M.L. and X.L. performed research; Z.W., Y.J., M.L. and X.L. analyzed data; W.J. and Z.W. wrote the paper.
Disclosure Statement The authors have no potential conflicts of interest to disclose.
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Pharmacology 2015;96:25–31 DOI: 10.1159/000431100
Ginsenoside Rg1 Ameliorates Motor Function in an Animal Model of Parkinson’s Disease Wenda Jiang a Zhejian Wang b Yiyang Jiang a Meili Lu c Xin Li d
a
Department of Neurology, First Affiliated Hospital of Liaoning Medical University, Jinzhou, b Dalian Medical University, Dalian, c Institute of Medicine, Liaoning Medical University, Jinzhou, d Department of Neurology, Jinzhou Central Hospital, Jinzhou, China
Key Words Parkinson’s disease · Ginsenoside Rg1 · Dopaminergic neuron · MPTP
significantly rescue the deficit of motor function in mice model of PD, and suggest that Rg1 may be a potential therapeutic agent against PD and related disorders. © 2015 S. Karger AG, Basel
© 2015 S. Karger AG, Basel 0031–7012/15/0962–0025$39.50/0 E-Mail [email protected] www.karger.com/pha
Introduction
Parkinson’s disease (PD) is one of the major neurodegenerative disorders, which is characterized by disabling motor abnormalities including tremor, muscle stiffness, paucity of voluntary movements and postural instability [1–3]. The main neuropathological feature of PD is the loss of dopaminergic neurons in the substantia nigra (SN) and diminished dopamine levels in the striatum. Accumulating evidence supports that nigral neuronal loss in PD is at least in part due to the aggregation of α-synuclein protein in the cytoplasm [4, 5]. Indeed, genetic studies have shown that α-synuclein knockdown by siRNAs protects dopaminergic neurons from MPTP-induced cell death both in vitro and in vivo [6–9]. Ginseng has been used in traditional Chinese medicine to enhance stamina and capacity to deal with fatigue and physical stress for thousands of years. Ginsenoside Rg1 is the major active ingredient of ginseng. It has been widely reported that Rg1 has a wide range of neurotrophic Wenda Jiang, MD Department of Neurology First Affiliated Hospital of Liaoning Medical University 5-2 Renmin Street, Jinzhou 121000 (China) E-Mail jiang_neurology @ 163.com
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Abstract Aims: Parkinson’s disease (PD) is a neurodegenerative disorder characterized by loss of dopaminergic neurons in the substantia nigra (SN) and diminished dopamine levels in the striatum. Accumulating evidence supported that ginsenoside Rg1, the major pharmacologically active compound of ginseng, has a wide range of neurotrophic and neuroprotective effects under physiological and pathological conditions. Although Rg1 administration protects dopaminergic neurons in a rat model of PD, it is unclear if Rg1 treatment ameliorates motor function in PD. Methods: Using the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) that causes dopaminergic neurodegeneration, we investigated the effect of Rg1 on 3 tests of motor behaviors in mice: the accelerating rotarod, wire suspension and pole tests. Results: The results showed that Rg1 treatment (10 mg/kg, i.p.) succeeded in restoring motor functions to physiological level in MPTP-treated mice. Importantly, these behavioral ameliorations were accompanied by an attenuation of the MPTP-induced loss of dopaminergic neurons in the SN and striatum. Conclusions: These findings indicate that Rg1 can
Rg1 injection
Fig. 1. Experimental design. Mice received
and neuroprotective effects both in vitro and in vivo. For example, in vitro studies have showed that Rg1 protects dopaminergic cells against glutamate [10], MPTP [11, 12] and rotenone toxicities [13]. In vivo studies have also demonstrated the protective effect of Rg1 against MPTPinduced nigral neuronal loss [14]. However, it is undetermined if Rg1 treatment ameliorates motor function in MPTP-treated model of PD. Since a growing body of evidence has showed that Rg1 has obviously protective effects on dopaminergic neurons, we hypothesize that Rg1 treatment may ameliorate motor function. In this study, we investigated this hypothesis using a combination of behavioral, immunoblotting and immunohistochemical assessments in MPTP-treated mice. Materials and Methods Animals and Treatment C57BL/6 mice (25–30 g, purchased from Charles River (Beijing Office, China)) were housed in plastic cages with free access to food and water and maintained in a temperature-controlled room (21 ° C) with a 12/12 h light/dark cycle. All behavioral experiments were performed according to the guidelines of Liaoning Medical University Animal House Center and every effort was made to minimize both the animal suffering and the number of animals used. Mice received intraperitoneal (i.p.) injection of 30 mg/kg parkinsonian toxin MPTP hydrochloride once a day for 5 days to induce dopaminergic neuron death in the SN, while the control mice received equal amount of saline injection. Rg1 (10 mg/kg, i.p.) was injected into the MPTP-treated mice once a day from the first day of MPTP injection until 10 days after the last injection of MPTP. The experimental design is described in fig. 1.
Chemicals and Reagents Anti-α-synuclein and anti-tyrosine hydroxylase (TH) antibody are obtained from BD Transduction Laboratories. Anti-β-actin antibody is obtained from Abcam. Rg1 and MPTP hydrochloride are obtained from Sigma. Rotarod Test One week after the last MPTP injection, mice performances on the rod were evaluated on the accelerating rotarod apparatus (Stoelting Co.). The test consisted of a suspended rod (diameter:
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MPTP injection
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3 cm) that accelerates at a constant rate, from 4 to 40 rpm in 300 s. Mice were tested 3 consecutive days and at each day of test, they were placed on the rod for a session of 10 trials at 3 min intervals, and the time that they remained on the rotarod during each trial was recorded. The maximum test time (cut-off limit) was 300 s. Pole Test The pole test was modified from previously reported protocols [15]. The pole consisted of a thin wooden cylinder (length: 50 cm, diameter: 1.5 cm) and a cross-shaped wooden base placed in a clean cage. Rubber bands were wrapped around the cylinder at intervals of approximately 1.5 inches to increase traction. Mice were acclimated to the testing room for 1 h before each session. Mice were placed at the top of the pole facing downwards and latency to descend the pole was measured. Trials were excluded if the mouse jumped or slid down the pole rather than climbed down. Mice were pretrained 2 consecutive times at the first day of test and the test was performed at days 1, 2 and 3 before the rotarod test. Wire Suspension Test A wire (length: 80 cm, height: 25 cm) was fixed horizontally between two platforms. Each animal was hung with its paws from the middle of the wire, and the time needed to reach one platform was recorded. The maximal time allowed was set at 120 s. Performance was conducted at days 1, 2 and 3 before the initiation of the rotarod test. Immunoblotting Brain tissues were lysed on ice in the lysis buffer and then the solution was centrifuged at 14,000 rpm for 10 min at 4 ° C. Supernatant was collected and protein concentration was determined by BCA protein assay kit (Thermo Scientific). An equal amount of protein samples were mixed with 4× sample buffer, boiled at 100 ° C for 5 min, and separated on 10% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Proteins were then transferred to Immobilon-PTM polyvynilidene fluoride (PVDF) membranes (Bio-Rad). The membranes were blocked with 5% non-fat milk in Tris-buffered saline containing 0.1% Tween-20 (TBST) for 1 h at room temperature, and then incubated overnight at 4 ° C with primary antibody. After washing 3 × 5 min in TBST, membranes were incubated with horseradish peroxidase-conjugated secondary antibody for 1 h at room temperature. After another three washes with TBST, protein was visualized in the BioRad Imager using ECL Western blotting substrate (Pierce). The band density of each protein was quantified by the Bio-Rad Quantity One software and the relative optical density was analyzed relative to loading control beta-actin on the same membrane.
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a chronic MPTP treatment consisting of 1 injection (i.p.) of 30 mg/kg/day or the same volume of sterile saline for 5 days, and Rg1 (10 mg/kg, i.p.) was administrated daily throughout the experiment. Seven days after the last injection of MPTP, behavioral tests were performed for 3 days, and brain tissues were collected immediately after behavioral tests.
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Fig. 2. Behavioral tests. Effects of Rg1 treatment on MPTP-induced motor deficits in the paradigms of the rotarod test (a), pole test (b) and wire suspension test (c). Statistical analyses were car-
ried out using the two-way ANOVA, Tukey’s post hoc analysis. # p < 0.05, ## p < 0.01 vs. respective first day; * p < 0.05 vs. respective saline-treated mice. n = 10 in each group.
Immunohistochemistry Mice were anesthetized with 1.5 g/kg urethane (Sigma) and then perfused with 0.9% saline and 4% paraformaldehyde. The brain was taken out from the mice and fixed in 4% paraformaldehyde for one more day before they were transferred to 30% sucrose/PBS solution for cryoprotection. After the brain slices had sunk to the bottom of the sucrose solution, they were sectioned into 30 μm slices using Leica cryostat. After blocking and permeabilization using PBS solution containing 1% BSA and 0.2% Triton X-100 for 30 min at room temperature, the brain slices were incubated with anti-tyrosine hydroxylase antibody (1: 800 dilution) for 3 days at 4 ° C. Finally, the tyrosine hydroxylase-positive neurons on the slices were stained and visualized under light microscope using the anti-mouse Ig HRP detection kit (BD Transduction Laboratories, 551011) according to the manufacturer’s instruction.
(fig. 2a), indicating an impairment of motor balance and coordination. Importantly, daily Rg1 treatment fully rescued the MPTP-induced motor deficit, as reflected by an increase in the latency to fall off on test days 2 and 3 (fig. 2a). To further investigate the influence of Rg1 on motor capacities of MPTP-treated mice, we next introduced another two behavioral tasks, pole test and wire suspension test. Pole test estimated bradykinesia and motor coordination, whereas wire suspension test analyzed the coordination and muscle strength of mice [16, 17]. The results from pole test showed that mice treated with MPTP spent much more time descending the pole compared with those treated with saline (fig. 2b). As expected, Rg1 treatment succeeded in rescuing the motor deficit induced by MPTP, as reflected by a similar time in descending the pole compared with saline control (fig. 2b). Similarly, in comparison with saline controls, mice treated with MPTP spent much more time reaching the platform during the wire suspension test (fig. 2c), and Rg1 treatment restored the motor capacity to control levels (fig. 2c). Taken together, these results indicate that MPTP treatment produces Parkinson-like motor deficits that are frequently observed in patients with PD, and chronic Rg1 treatment can dramatically rescues these deficits.
Statistical Analysis Animals were randomly chosen for experiments and the data were analyzed by SPSS 13.0 and expressed as mean ± SEM. The differences of motor performance among different groups of mice were analyzed by two-way ANOVA, followed by Tukey’s post hoc test. The data of all other experiments were analyzed by one-way ANOVA, followed by Tukey’s post hoc test. * p < 0.05 and ** p < 0.01 were considered significant differences.
Results
Rg1 Ameliorates MPTP-Induced Motor Dysfunction Previous studies have shown that a core symptom of PD is motor abnormality [1–3]. To determine whether Rg1 can rescue MPTP-induced motor deficit, we first used a rotarod test to evaluate the motor balance in mice treated with Rg1. The results showed that the mice treated with MPTP spent significantly less time on the rod on test days 2 and 3 compared with those treated with saline Rg1 Treats Parkinson’s Disease
Rg1 Attenuates MPTP-Induced Loss of Dopaminergic Neurons in the SN and Striatum It is well documented that MPTP treatment produces loss of dopaminergic neurons in both SN and striatum [18–20]. To evaluate the protective effect of Rg1 on dopaminergic neurons, we examined the level of TH that is Pharmacology 2015;96:25–31 DOI: 10.1159/000431100
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known to correlate with the level of dopaminergic neurons [21, 22]. As revealed by previous reports [18–20], we observed that TH expression significantly decreased in mice treated with MPTP in both SN (fig. 3a and b) and striatum (67.6 ± 10.1% saline, p < 0.05; fig. 3d and e) compared with saline control. Consistent with a recent report that Rg1 attenuates MPTP-induced apoptosis in mouse SN neurons [14], we found that chronic Rg1 treatment dramatically displayed the protective effect on loss of dopaminergic neurons in both SN (fig. 3a and b) and striatum (fig. 3d and e). Since previous reports have supported that loss of dopaminergic neurons in PD is at least in part due to the aggregation of α-synuclein protein in the cytoplasm [4, 5], we next examined if Rg1 treatment resulted in the degradation of α-synuclein in both SN and striatum. The result showed that MPTP treatment inPharmacology 2015;96:25–31 DOI: 10.1159/000431100
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duced a significant increase in α-synuclein expression in both SN (fig. 3a and c) and striatum (fig. 3d and f) compared with saline control. As expected, Rg1 treatment succeeded in restoring the α-synuclein expression to control level in both SN (fig. 3a and c) and striatum (fig. 3d and f). In order to further evaluate the protective effect of Rg1 on dopaminergic neurons, we counted TH-positive neurons in both SN and striatum. As showed in figure 4, dopaminergic neurodegeneration occurred morphologically after MPTP treatment. The numbers of TH-positive neurons significantly decreased in both SN (fig. 4a) and striatum (fig. 4b) compared with saline control. Similar to the results shown in figure 3, Rg1 treatment rescued the loss of dopaminergic neurons in both SN (fig. 4a) and striatum (fig. 4b). Jiang/Wang/Jiang/Lu/Li
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Fig. 3. Expression levels of TH and α-synuclein proteins were assessed by the Western blotting assay. a–c Immunoblotting for TH and α-synuclein in the SN. d–f Immunoblotting for TH and α-synuclein in the striatum. Statistical analyses were carried out using the one-way ANOVA, Tukey’s post hoc analysis. * p < 0.05 vs. saline-treated mice. n = 5 in each group.
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Discussion
In this study, we confirmed that ginsenoside Rg1, one of the active ingredients of ginseng, reduced the loss of dopaminergic neurons in both SN and striatum, and demonstrated that chronic Rg1 treatment ameliorated motor function and increased α-synuclein degradation in the MPTP-induced mice model of PD. Together, these results indicated that the neuroprotective mechanism of Rg1 may be attributed to an increase in the degradation of α-synuclein and subsequently to the prevention of MPTP-induced loss of dopaminergic neurons in SN and striatum. We have therefore provided evidence that Rg1 may be a potential therapeutic agent against PD via degrading increased α-synuclein in the SN and striatum. Rg1 Treats Parkinson’s Disease
0.3
The pathological hallmark of PD is the loss of dopaminergic neurons in the SN and diminished dopamine level in the striatum. Consistent with previous studies [18], we here found that dopaminergic neurons dramatically reduced in both SN and striatum in the MPTP-induced mice model of PD. But molecular mechanism underlying neuronal loss is not clear. Recent studies have shown that the abnormal accumulation of α-synuclein in the brain is a common feature of PD [23], and this may contribute to nigral neuronal loss in PD [4, 5]. Thus, it is reasonable to propose that the degradation of MPTP-induced α-synuclein may reduce the loss of dopaminergic neurons and subsequently rescue motor deficits in the MPTP-induced mice model of PD. Indeed, this hypothesis is supported by some previous genetic studies. For instance, knockdown α-synuclein by siRNAs protects dopaminergic Pharmacology 2015;96:25–31 DOI: 10.1159/000431100
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sentative images (left panel) and bar graph (right panel) show the influence of Rg1 on MPTP-induced the loss of TH-positive neurons in the SN. b Representative images (left panel) and bar graph (right panel) show the influence of Rg1 on MPTP-induced the loss of striatal fibers. Statistical analyses were carried out using the oneway ANOVA, Tukey’s post hoc analysis. * p < 0.05, ** p < 0.01 vs. saline-treated mice. n = 8 in each group.
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neurons from MPTP-induced cell death both in vitro and in vivo [6–9]. In full agreement with these findings, we here reported that chronic Rg1 obviously increased the degradation of α-synuclein, and reduced the loss of dopaminergic neurons in the SN and striatum as well as rescued motor deficits. However, the involvement of Rg1 in α-synuclein degradation has been challenged, and a recent report indicates that only Rb1, but not Rg1 and Rg3, exhibits an obvious ability to disaggregate preformed fibrils of α-synuclein [24]. Discrepancies between the previous study and the present study still need to be resolved but may be at least in part accounted for by different experimental conditions, such as in vitro or in vivo. Alternatively, the neuroprotective effect of Rg1 against MPTP-induced neuronal loss may be related to the enhanced expression of Bcl-2 and reduced expression of nitric oxide synthase (NOS). It has been reported that Bcl-2 protects cells from apoptosis induced by exogenous oxidants or conditions that increase their intracellular production and at the same time reduce oxidative damage to cellular constituents [25–27]. Recent reports further confirmed the view that Rg1 partially overcame the decrease of Bcl-2 protein and gene expressions induced by 6-OHDA [28] or MPTP [14], therefore positively regulating cell survival. However, the exact pathway leading to the changes of Bcl-2 and NOS expression in the SN and
striatum is still unclear. Thus, future experiments determining the Bcl-2- and/or NOS-related signal pathways affected by Rg1 will be helpful to clinical application of Rg1 in treatment with PD and related disorders.
Conclusions
In summary, chronic ginsenoside Rg1 treatment reduces the loss of dopaminergic neurons in both SN and striatum and ameliorates motor function in the MPTPinduced mice model of PD. These ameliorations were accompanied by an increase in α-synuclein degradation. Thus, our findings indicate that Rg1 may be a potential therapeutic agent against PD in the future.
Acknowledgments W.J. designed research; W.J., Z.W., Y.J., M.L. and X.L. performed research; Z.W., Y.J., M.L. and X.L. analyzed data; W.J. and Z.W. wrote the paper.
Disclosure Statement The authors have no potential conflicts of interest to disclose.
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